Inflammatory processes are characterized by an increase in microvascular permeability (hypermeability) to macromolecules. Signaling interactions between vascular wall and blood cells provide a unique way of communicating, coordinating, and integrating an appropriate physiologic response to the changing tissue environment in vivo. There is a paucity of information on permeability-related signaling mechanisms in the complex cellular environment of the in vivo microcirculation. Knowledge of these important interactions in vivo is fundamental to understand the integrated regulation of microvascular transport and its functional alterations in vascular disease. In particular, the role of nitric oxide (NO) in the control of microvascular permeability remains controversial. We propose to test in vivo three major hypotheses: 1) an eNOS-assoctated signaling cascade regulates microvascular transport. 2) molecular movement (translocation, trafficking) of eNOS from membrane to other cellular compartments is a functionally and differentially important step in the endothelium-mediated regulatory mechanisms. 3) eNOS signaling mechanisms regulate microvascular permeability responses to ischemia-reperfusion (I-R). To test these hypotheses, we will A) determine whether or not a cause-effect relationship exists between eNOS activity and hyperpermeability in vivo in eNOS -/- mice and their wild-type eNOS +/+ control; B) test translocation of eNOS from membrane to cytosol after stimulation with a pure vasodilator (ACh) and a non-vasodilator that causes hyperpermeability (PAF); C) determine whether or not differences in microvascular permeability responses exist in eNOS +/+ and eNOS -/- mice and the subcellular location of eNOS under baseline and after I-R. In addition, we will determine whether or not changes in eNOS phosphorylation occur in response ACh, PAF and to I-R, and assess the significance of PKB and MAP kinases in these processes. Methods of immunoprecipitation, western blotting, intravital and confocal microscopy, as well as NO measurements and computer-assisted digital image analysis will be applied to evaluate the regulation of microvascular permeability in vivo.